Actin cytoskeleton plays an important role in differentiated vascular smooth muscle cells (dVSMCs) because they maintain and change the cell shape in concert with the activities of the contractile apparatus. A large number of actin-binding proteins (ABPs) participate in these processes, many of which contain multiple binding modules and are subject to phosphorylation. The underlying hypothesis of Project 2 is that phosphorylation alters the actin-binding properties of ABPs as a consequence of cell signaling, and thereby allows for remodeling of the actin cytoskeleton. This hypothesis will be tested the mechanistic basis for such remodeling and its regulation in dVSMCs will be investigated. With previously demonstrated approaches, the phosphorylation-dependent conformational changes will be determined using five selected cytoskeleton proteins (caldesmon, calponin, cortactin, VASP and zyxin) as model cases. The structural information thus obtained will be compared to the results from Projects 3 and 4. A second hypothesis that cytoskeleton proteins work together as partners to synergistically maintain the actin cytoskeleton structures will also be tested. In support of this idea a recently developed mouse model in which the smooth muscle caldesmon gene is disrupted, demonstrated that a number of cytoskeleton proteins exhibited decreased levels of expression along with the elimination of smooth muscle caldesmon. The potential interactions between caldesmon and these proteins will be examined. Novel biophysical tools such as surface plasmon resonance and isothermal titration calorimetry will be used. In addition, the sites of interaction will be determined and synthetic peptides or recombinant fragments will be prepared corresponding to the binding sequences. These peptides/fragments will then be used as decoys to test the functional significance of the interactions and phosphorylation in primary SMCs and in isolated dVSMCs. The localization of cytoskeleton proteins as well as the morphology of the cytoskeleton structure will be examined by immuno-fluorescence and immuno-electron microscopy, together with the findings from Project 1, to gain insights into the effects on smooth muscle contractility. Since the contraction of vascular smooth muscles controls the blood pressure in our body, mulfunction of vascular smooth muscle leads to hypertension and other cardiovascular diseases. Information obtained from this study will help develop therapeutic reagents for these diseases.